1. Field of the Invention
The present invention relates generally to the field of harmonic generation microscopy, and in particular to a microscopic imaging technique using both second and third harmonic waves of a excitation spectrum of a laser beam by a sample to form an image of the sample.
2. The Related Art
Microscopic imaging has been widely used in a variety of applications. For example, microscopic observation of a biological tissue is one of the best-known applications of the microscopic imaging techniques. The recent development of the microscopic imaging allows for the employment of laser beam in high precision observation of biological samples. An example of the laser-based microscopy is two-photon laser scanning fluorescent microscopy that was published in Science, New Series, Volume 248, Issue 4951 (Apr. 6, 1990), pp 73-76, by Winfried Denk, James H. Strickler and Watt W. Webb. A biological sample is stained by a fluorescent dye. The fluorescent dye molecule is excited by simultaneously absorbing two photons of the same wavelength to give off fluorescent light, which is received and processed for imaging of the biological sample. In this respect, this technique is also applicable to autofluorescent biological samples. The image obtained with the two-photon fluorescent microscopy has excellent resolution. However, the fluorescent dye may be toxic to in vivo samples. Further, the excitation of the fluorescent light by absorption of photons may induce photo-damages to the observed samples. For example, with a Ti: Sapphire laser having a pulse duration 100 fs and a repetition rate of 80 MHz, an average power exceeding 6 mW causes photo-damage to the samples.
Contrary to the fluorescent microscopy, a harmonic generation microscopy induces less photo-damage to the samples. The most commonly known harmonic generation microscopy includes second harmonic generation (SHG) microscopy and third harmonic generation (THG) microscopy.
SHG that was originally employed in the research of second harmonic generation crystals has recently been used in the observation of noncentrosymmetric biological samples, such as “Second-Harmonic Imaging in the Scanning Optical Microscope” by J. N. Gannaway and C. J. R. Sheppard (1978), Optic Quantum Electron, Volume 10, pp. 435-439. SHG often occurs in a noncentrosymmetric and continuous structured media, especially nano-structures, such as stacked membranes, aligned protein structures, and microtubule arrays. However, SHG is not suitable for the observation of interfaces in biological samples.
THG microscopy was first published in Applied Physics Letters, 1997, Volume 70, pp. 922-924 by Y. Barad, H. Eisenberg, M. Horowltz and Y. Silberberg, which is employed in the observation of transparent media by means of the third harmonic generation occurring in the interface. Since all materials have non-varnishing third order coefficient and since the coefficient is different at different portions of an observed sample, which induces variation of THG intensity, the THG microscopy is commonly used in non-linear scanning microscopic imaging process. Since THG often occurs in the interface, THG is not suitable for observation of bulk noncentrosymmetric media, which, however, can be clearly inspected by means of SHG.
An example of the THG microscopy is disclosed in U.S. Pat. No. 5,828,459.
A similar technique is disclosed in U.S. Pat. No. 6,208,886, which uses a laser source, such as Ti: Sapphire laser, Cr: Forsterite laser and Nd: Yag laser, to issue a laser beam having a wavelength within the range of 400-1400 nm. The radiation excites the observed sample to give off fluorescent light and third harmonic wave. Due to the generation of the fluorescent light, photo-damage is inherent in this technique. Further, due to lacking of the second harmonic wave, noncentrosymmetric object cannot be well examined by this technique.
Apparently, the conventional microscopic imaging techniques discussed above have one or more drawbacks in providing clear and wide range observation of a sample.